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Oligonucleotide Properties Calculator

Enter Oligonucleotide Sequence Below
OD and Molecular Weight calculations are for single-stranded DNA or RNA

Reverse Complement Strand(5' to 3') is:

Number of Fluorescent tags per strand:

6-FAM	TET	HEX	TAMRA
Minimum base pairs required for single primer self-dimerization:
Minimum base pairs required for a hairpin :

Physical Constants

Melting Temperature (T_M) Calculations

Length: bases
GC content: %
Molecular Weight: ⁴
1 ml of a sol'n with an Absorbance of at 260 nm
is microMolar ⁵ and contains micrograms.

1 °C (Basic)
2 °C (Salt Adjusted)
3 °C (Nearest Neighbor)
nM Primer
mM Salt (Na⁺)

Thermodynamic Constants
Conditions: 1 M NaCl at 25°C at pH 7.

RlnK cal/(°K*mol)

deltaH Kcal/mol

deltaG Kcal/mol

deltaS cal/(°K*mol)

To use this calculator, you must be using Netscape 3.0 or later
or Internet Explorer version 3.0 or later, or another Javascript-capable browser
Self-Complementarity requires a 4.x browser. IE 5.0, Safari, and Mozilla supported.
This page was written in Javascript.
Extensively rewritten from 12/15/2000-12/19/2000 to isolate javascript Oligo object behaviors for teaching purposes.
This page may be freely distributed for any educational or non-commercial use.
Copyright Northwestern University, 1997-2004.
Version history

About the Calculations

Thermodynamic Calculations

The nearest neighbor and thermodynamic calculations are done essentially as described by Breslauer et al., (1986) Proc. Nat. Acad. Sci. 83:3746-50 (Abstract) but using the values published by Sugimoto et al., (1996) Nucl. Acids Res. 24:4501-4505 (Abstract). RNA thermodynamic properties were taken from Xia T., SantaLucia J., Burkard M.E., Kierzek R., Schroeder S.J., Jiao X., Cox C., Turner D.H. (1998) Biochemistry 37:14719-14735 (Abstract). This program assumes that the sequences are not symmetric and contain at least one G or C. The minimum length for the query sequence is 8.

The melting temperature calculations are based on the simple thermodynamic relationship between entropy, enthalpy, free energy and temperature, where

The change in entropy (order or a measure of the randomness of the oligonucleotide) and enthalpy (heat released or absorbed by the oligonucleotide) are directly calculated by summing the values for nucleotide pairs obtained by Breslauer et al., Proc. Nat. Acad. Sci. 83, 3746-50, 1986. The relationship between the free energy and the concentration of reactants and products at equilibrium is given by

Substituting the two equations gives us

and solving for temperature T gives

We can assume that the concentration of DNA and the concentration of the DNA-primer complex are equal, so this simplifies the equation considerably. It has been determined empirically that there is a 5 (3.4 by Sugimoto et al.) kcal free energy change during the transition from single stranded to B-form DNA. This is presumably a helix initiation energy. Finally, adding an adjustment for salt gives the equation that the Oligo Calculator uses:

No adjustment constant for salt concentration is needed, since the various parameters were determined at 1 Molar NaCl, and the log of 1 is zero.

ASSUMPTIONS:
The thermodynamic calculations assume that the annealing occurs at pH 7.0. The melting temperature (Tm) calculations assume the sequences are not symmetric and contain at least one G or C. The oligonucleotide sequence should be at least 8 bases long to give reasonable Tms.

Basic Melting Temperature (Tm) Calculations

The two standard approximation calculations are used. For sequences less than 14 nucleotides the formula is

Tm= (wA+xT) * 2 + (yG+zC) * 4

where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively.

For sequences longer than 13 nucleotides, the equation used is

Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)

ASSUMPTIONS:
Both equations assume that the annealing occurs under the standard conditions of 50 nM primer, 50 mM Na⁺, and pH 7.0.

Salt Adjusted Melting Temperature (Tm) Calculations

A variation on two standard approximation calculations are used. For sequences less than 14 nucleotides the same formula as the basic calculation is use, with a salt concentration adjustment

Tm= (wA+xT)*2 + (yG+zC)*4 - 16.6*log₁₀(0.050) + 16.6*log₁₀([Na⁺])

where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively.

The term 16.6*log₁₀([Na⁺]) adjusts the Tm for changes in the salt concentration, and the term log₁₀(0.050) adjusts for the salt adjustment at 50 mM Na⁺. Other monovalent and divalent salts will have an effect on the Tm of the oligonucleotide, but sodium ions are much more effective at forming salt bridges between DNA strands and therefore have the greatest effect in stabilizing double-stranded DNA.
For sequences longer than 13 nucleotides, the equation used is

Tm= 100.5 + (41 * (yG+zC)/(wA+xT+yG+zC)) - (820/(wA+xT+yG+zC)) + 16.6*log₁₀([Na⁺])

For oligos longer than 50, the following equation may be more accurate. However, for oligos in the 18-25mer range the above equation (which is what OligoCalc uses) is more accurate.

Tm= 81.5 + (41 * (yG+zC)/(wA+xT+yG+zC)) - (500/(wA+xT+yG+zC)) + 16.6*log₁₀([Na⁺]) - 0.62F

This equation is most accurate for sequences longer than 50 nucleotides. It is valid for oligos longer than 50 nucleotides from pH 5 to 9. Symbols and salt adjustment term as above, with the term (41 * (yG + zC-16.4)/(wA + xT + yG + zC)) adjusting for G/C content and the term (500/(wA + xT + yG + zC)) adjusting for the length of the sequence, and F is the percent concentration of formamide.

For more information please see the reference:
Howley, P.M; Israel, M.F.; Law, M-F.; and M.A. Martin "A rapid method for detecting and mapping homology between heterologous DNAs. Evaluation of polyomavirus genomes." J. Biol. Chem. 254, 4876-4883, 1979.

RNA melting temperatures

Tm= 79.8 + 18.5*log₁₀([Na⁺]) + (58.4 * (yG+zC)/(wA+xT+yG+zC)) + (11.8 * ((yG+zC)/(wA+xT+yG+zC))²) - (820/(wA+xT+yG+zC))

Where yG+zC are the mole fractions of G and C in the oligo, L is the length of the shortest strand in the duplex.

ASSUMPTIONS:
These equations assume that the annealing occurs under the standard conditions of 50 nM primer and pH 7.0.

Molecular Weight Calculations

DNA Molecular Weight (for instance synthesized Oligonucleotides)

Anhydrous Molecular Weight = (A_n x 313.21) + (T_n x 304.2) + (C_n x 289.18) + (G_n x 329.21) - 61.96

A_n, T_n, C_n, and G_n are the number of each respective nucleotide within the polynucleotide. The subtraction of 61.96 gm/mole from the oligonucleotide molecular weight takes into account the removal of HPO₂ (63.98) and the addition of two hydrogens (2.02).

Please note: this calculation works well for synthesized oligonucleotides. If you would like an accurate MW for restriction enzyme cut DNA, please use:

Molecular Weight = (A_n x 313.21) + (T_n x 304.2) + (C_n x 289.18) + (G_n x 329.21) + 79.0

The addition of 79.0 gm/mole to the oligonucleotide molecular weight takes into account the 5' monophosphate left by most restriction enzymes. No phosphate is present at the 5' end of strands made by primer extension, so no adjustment should be necessary.

RNA Molecular Weight (for instance from an RNA transcript)
Molecular Weight = (A_n x 329.21) + (U_n x 306.17) + (C_n x 305.18) + (G_n x 345.21) + 159.0
A_n, U_n, C_n, and G_n are the number of each respective nucleotide within the polynucleotide. Addition of 159.0 gm/mole to the molecular weight takes into account the 5' triphosphate.

OD Calculations

Molar Absorptivity values in 1/(Moles cm)

Residue	Moles^-1 cm^-1	A_max(nm)	Molecular Weight (after protecting groups are removed)
Adenine (dAMP, Na salt)	15200	259	313.21
Guanine (dGMP, Na salt)	12010	253	329.21
Cytosine (dCMP, Na salt)	7050	271	289.18
Thymidine (dTMP, Na salt)	8400	267	304.2
RNA nucleotides
Adenine (AMP, Na salt)	15400	259	329.21
Guanine (GMP, Na salt)	13700	253	345.21
Cytosine (CMP, Na salt)	9000	271	305.18
Uradine (UMP, Na salt)	10000	262	306.2
Other nucleotides
6' FAM	20960		537.46
TET	16255		675.24
HEX	31580		744.13
TAMRA	31980

Assume 1 OD of a standard 1ml solution, measured in a cuvette with a 1 cm pathlength.

6-FAM:

Chemical name:	6-carboxyfluorescein
Absorption wavelength maximum:	495 nm
Emission wavelength maximum:	521 nm
Molar Absorptivity at 260nm:	20960 Moles^-1 cm^-1

TET:

Chemical name:	4, 7, 2', 7'-Tetrachloro-6-carboxyfluorescein
Absorption wavelength maximum:	519 nm
Emission wavelength maximum:	539 nm
Molar Absorptivity at 260nm:	16255 Moles^-1 cm^-1

HEX:

Chemical name:	4, 7, 2', 4', 5', 7'-Hexachloro-6-carboxyfluorescein
Absorption wavelength maximum:	537 nm
Emission wavelength maximum:	556 nm
Molar Absorptivity at 260nm:	31580 Moles^-1 cm^-1

TAMRA:

Chemical name:	N, N, N', N'-tetramethyl-6-carboxyrhodamine
Absorption wavelength maximum:	555 nm
Emission wavelength maximum:	580 nm
Molar Absorptivity at 260nm:	31980 Moles^-1 cm^-1

Nucleotide base codes (IUPAC)

Symbol: nucleotide(s)

A adenine

C cytosine

G guanine

T thymine in DNA;
uracil in RNA

N A or C or G or T

M A or C

R A or G

W A or T

S C or G

Y C or T

K G or T

V A or C or G; not T

H A or C or T; not G

D A or G or T; not C

B C or G or T; not A

Most recent version is available at URL: http://www.basic.northwestern.edu/biotools/oligocalc.html

The current version is the result of efforts by the following people:

Qing Cao, M.S.
Research Computing
Northwestern University Medical School
Chicago, IL 60611

Warren A. Kibbe, Ph.D. and PH entry.
Research Computing
Northwestern University Medical School
Chicago, IL 60611

Original code by Eugen Buehler
Research Support Facilities
Department of Molecular Genetics and Biochemistry
University of Pittsburgh School of Medicine

Monomer structures and molecular weights provided by Bob Somers, Ph.D.

Sr. Applications Chemist
Glen Research Corporation
22825 Davis Drive
Sterling, VA 20164
http://www.glenres.com/

Uppercase/lowercase strand complementation problem described by Alexey Merz alexey@dartmouth.edu

The RNA calculations and functions were requested by Suzanne Kennedy Suzanne.Kennedy@qiagen.com